Plants rely on an internal clock in order to regulate the activity of a variety of internal biochemical and physiological processes. This clock is called the circadian rhythm, an important characteristic of which is its cyclical nature. Thus, physiological and biochemical processes regulated by the circadian clock are repeated in cycles, and each cycle is approximately 24 hours long. Molecular studies have revealed that the circadian rhythm is regulated by gene expression and its cyclical nature is due to the activity of autoregulatory negative feedback loops in which one or more gene products repress their own expression.
In addition to the circadian rhythm, plants also rely on external environmental cues, called Zeitgebers, such as light-dark cycles and changes in temperature or humidity, in order to regulate critical developmental processes, for example growth and freezing tolerance. This ability enables plants to acclimatize to a diverse range of climates and environments. Research has revealed that there is an interplay between a plant's circadian rhythm and its ability to respond to environmental cues (i.e. Zeitgebers). Indeed, environmental cues can be used to entrain (i.e. modulate) the length of a plant's circadian rhythm, and the biochemical and physiological processes which they regulate. Therefore, a plant's phenotype may be altered if it is exposed to certain artificial environmental cues.
Lignin is an organic substance responsible for binding cells, fibres and vessels of wood and herbaceous plants. Due to the limited supply of fossil fuels and the increasing demand for alternative forms of energy, lignin is becoming an increasingly important renewable energy resource. However, as lignin is present in growing plants, it helps to preserve the earth's atmosphere by acting as a natural reservoir for greenhouse gases. Accordingly, in order to maintain the balance between the preservation of the earth's environment, and replenishment of lignin, which is used as an energy resource, only a limited number of plants should be harvested for their lignin each year. Unfortunately, this is made difficult by the sub-optimal growth conditions to which plants in a large proportion of the world's forests are exposed. Indeed, trees grown in such sub-optimal conditions tend not to develop fully due to extreme seasonal variations in day length, and so their lignin concentrations are lower than plants grown in standard conditions. There is therefore a need to increase the lignin content of plants which are used as a renewable energy resource, especially plants which are usually exposed to poor growth conditions.
Lignin is also a prominent constituent of tobacco plants, the leaves of which are commonly used in smoking articles, such as cigarettes and cigars. When combusted, the lignin within cured tobacco leaves, undergoes a process called pyrolysis, and releases natural chemicals which may be inhaled during the smoking process. Some of these released chemicals contribute to the smoky flavour produced during the smoking process. While some users may find that these chemicals enhance the smoking experience, others can find that they can compromise the flavour of the inhaled smoke. Accordingly, there is also a need to reduce the lignin content of plants, and particularly their leaves, which are smoked, such as tobacco.
As described in the Examples, the inventors set out to investigate if there was a link between the circadian rhythm and the density of lignin within various plant species, with the aim of controlling plant lignin density. Their research prompted them to create two transgenic Poplar plant line cell lines, the first of which exhibited reduced expression of the core clock genes, LHY1 and LHY2, compared to its wild-type counterpart grown under the same conditions, and the second of which exhibited an enhanced expression of the core clock genes compared to its wild-type counterpart also grown under the same conditions. Global gene expression, growth and lignin density, among other characteristics of these transgenic plants, were then assessed.